Small RNAs are considered to play a very important role in regulation of chromatin structure, chromosome segregation, transcription, RNA processing, RNA stability, and translation. RNAs extracted from eukaryotic cells are classified into five different categories: ribosomal RNAs (rRNA), transfer RNAs (tRNA), messenger RNAs (mRNAs), long noncoding RNAs, and small RNAs. Over 90% of the total RNA molecules in the extract are rRNA and tRNA. Small RNAs account only for about 1% of the total RNA population, this include microRNAs (miRNAs), small interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs).
High-throughput sequencing (HTS, or next generation sequencing) techniques such as Illumia®, SOLiD® (Sequencing by Oligonucleotide Ligation and Detection) and Roche 454 pyrosequencing, are playing very important role in novel small RNA detection and miRNA editing detection.
In order to generate RNA sequencing libraries, the isolated native RNA is usually reverse transcribed into DNA followed by PCR-amplification. The unknown RNAs therefore need to be ligated to a known sequence of two adaptors or handles, one at the 3′-end and the other at the 5′-end of the native RNA. The 3′-handle installation is often done either by poly-A tailing or by 3′-adaptor ligation catalyzed by a T4 RNA ligase. As small RNAs are just over 20 bases long, poly-A tailing will generate a great percentage of poly-A signal. It also has the disadvantage of the uncertainty about the origin of the poly-A region and is often followed by an additional parallel poly C tailing reaction, which further complicates the procedure. This technique is considered to be not suitable for small RNA sequencing. So the 3′-handle is normally provided by a 3′-adaptor.
The 3′-adaptor ligation and the 5′-adaptor ligation reactions need to be separated by a gel purification step, otherwise the excessive 3′-adaptor will ligate to the 5′-adaptor.
Sequencing bias of the small RNA profiling result generated by HTS has been reported and systematically studied. This bias, occurs because some target RNAs in the sample adopt secondary structure in presence of the ligation buffer, leading to different adaptor ligation efficiency. This results an uneven representation of the RNA population in the sequencing library. The bias affects the reliability of studies to measure the expression level of miRNAs. The 3′-adaptor ligation step conducted by T4 RNA ligase is the cause of major RNA sequencing bias.
Despite the drawback of using ligase in RNA sequencing methods, no practical alternative has replaced its use and practical methods of mitigating the ligation-mediated sequence bias are continually sought.